Many metals pose a risk as environmental contaminants. A well-known example is lead. Low level lead exposure can lead to a number of adverse health effects, with as many as 9-25% of pre-school children presently at risk. The level of lead in the blood considered toxic is ≧10 μg/dL (480 nM). Current methods for lead analysis, such as atomic absorption spectrometry, inductively coupled plasma mass spectrometry, and anodic stripping voltammetry, often require sophisticated equipment, sample pre-treatment, and skilled operators.
Simple, rapid, inexpensive, selective and sensitive methods that permit real time detection of Pb2+ and other metal ions are very important in fields such as environmental monitoring, clinical toxicology, wastewater treatment, and industrial process monitoring. Furthermore, methods are needed for monitoring free or bioavailable, instead of total, metal ions in industrial and biological systems.
Many fluorescent chemosensors, including fluorophore-labeled organic chelators (Rurack, et al., 2000; Hennrich et al., 1999; Winkler et al., 1998; Oehme & Wolfbeis, 1997) and peptides (Walkup & Imperiali, 1996; Deo & Godwin, 2000; Pearce et al., 1998), have been developed for metal ion detection. These ion sensors are usually composed of an ion-binding motif and a fluorophore. Metal detection using these fluorescent chemosensors relies on the modulation of the fluorescent properties of the fluorophore by the metal-binding event. Detection limits on the level of micromolar and even nanomolar concentrations have been achieved for heavy metal ions including Zn2+, Cu2+, Hg2+, Cd2+ and Ag+. The design and synthesis of a chemosensor that exhibits highly selective and sensitive binding of the metal ion of choice in aqueous solution is still a big challenge, although the metal binding and the fluorescent moieties of the sensor can be systematically varied to achieve desired properties. Although fluorescence spectroscopy is a technique well suited for detecting very small concentrations of analytes, a fluorometer is required to generate and detect the emitted signal. Thus, the need for expensive instrumentation and complicated operation procedures make this method impractical for applications such as household use, field testing or small clinic testing.
Nucleic acid molecules have previously been adapted to sense the presence of nucleic acids and to detect gene mutations from inherited diseases or chemical damages. In recent years, the molecular recognition and catalytic function of nucleic acids have been extensively explored. This exploration has lead to the development of aptamers and nucleic acid enzymes.
Aptamers are single-stranded oligonucleotides derived from an in vitro evolution protocol called systematic evolution of ligands by exponential enrichment (SELEX). Nucleic acid aptamers have been isolated from random sequence pools and can selectively bind to non-nucleic acid targets, such as small organic molecules or proteins, with affinities as high as 10−14 M (Uphoff et al., 1996; Famulok, 1999). Most aptamers undergo a conformational change when binding their cognate ligands. With this property, several DNA and RNA aptamers have been engineered to sense L-adenosine or thrombin through an internally labeled fluorescent reporter group (Jhaveri et al., 2000). Here, the conformational change in the aptamer upon binding leads to a change in fluorescence.
Nucleic acid enzymes are nucleic acid molecules that catalyze a chemical reaction. In vitro selection of nucleic acid enzymes from a library of 1014-1015 random nucleic acid sequences offers considerable opportunity for developing enzymes with desired characteristics (Breaker & Joyce, 1994; Breaker, 1997). Compared with combinatorial searches of chemo- and peptidyl-sensors, in vitro selection of DNA/RNA is capable of sampling a larger pool of sequences, amplifying the desired sequences by polymerase chain reactions (PCR), and introducing mutations to improve performance by mutagenic PCR.
Allosteric ribozymes (or aptazymes), which combine the features of both aptamer and catalytic RNA, also hold promises for sensing small molecules (Potyrailo et al., 1998; Koizumi et al., 1999; Robertson & Ellington, 1999, 2000). Their reactivity is modulated through the conformational changes caused by the binding of small organic molecules to an allosteric aptamer domain. Therefore, the signal of ligand binding can be transformed into a signal related to chemical reaction.
Divalent metal ions can be considered as a special class of cofactors controlling the activity of nucleic acid enzymes. The reaction rate of the nucleic acid enzymes depends on the type and concentration of the metal ion in solution. Several RNA and DNA enzymes obtained through in vitro selection are highly specific for Cu2+, Zn2+, and Pb2+, with metal ion requirements on the level of micromolar concentrations (Breaker & Joyce, 1994; Pan & Uhlenbeck, 1992; Carmi et al., 1996; Pan et al., 1994; Cuenoud & Szotak, 1995; Li et al., 2000; Santoro et al., 2000).
A variety of methods have been developed for assembling metal and semiconductor colloids into nanomaterials. These methods have focused on the use of covalent linker molecules that possess functionalities at opposing ends with chemical affinities for the colloids of interest. One of the most successful approaches to date, (Brust et al., (1995)), involves the use of gold colloids and well-established thiol adsorption chemistry (Bain & Whitesides, (1989); Dubois & Nuzzo (1992)). In this approach, linear alkanedithiols are used as the particle linker molecules. The thiol groups at each end of the linker molecule covalently attach themselves to the colloidal particles to form aggregate structures. The drawbacks of this method are that the process is difficult to control and the assemblies are formed irreversibly. Methods for systematically controlling the assembly process are needed if the materials properties of these structures are to be exploited fully.
The potential utility of DNA for the preparation of biomaterials and in nanofabrication methods has been recognized. Researchers have focused on using the sequence-specific molecular recognition properties of oligonucleotides to design impressive structures with well-defined geometric shapes and sizes. Shekhtman et al., (1993); Shaw & Wang, (1993); Chen et al., (1989); Chen & Seeman, (1991); Smith and Feigon (1992); Wang et al., (1993); Chen et al., (1994); Marsh et al., (1995); Mirkin (1994); Wells (1988); Wang et al., (1991). However, the theory of producing DNA structures is well ahead of experimental confirmation. Seeman et al., New J. Chem., 17, 739-755 (1993).
Agglutation assays are well known for the detection of various analytes. The basic principle of an agglutination assay is the formation of clumps (agglutination or aggregation) of small particles coated with a binding reagent when exposed to a multi-valent binding partner specific for the binding reagent. Particles routinely used in agglutation assays include, for example, latex particles, erythrocytes (RBCs), or bacterial cells (often stained to make the clumps visible). Typically, a binding reagent, such as an antibody, is attached to the particles. A sample thought to contain the analyte of interest is contacted with a suspension of such coated particles. If the analyte is present, cross linking of the particles occurs due to bond formation between the antibodies on the particles and the analyte in the sample. Such binding results in the agglutation of the particles which can be detected either visually or with the aid of simple instrumentation. In an alternative protocol, an antigen is attached to the particles and the presence of an antibody specific for the antigen detected in the sample.
More recently, metal, semiconductor and magnetic particles have been used in aggregation assays. For example, particles comprising metal (e.g., gold, silver, copper and platinum), semiconductor (e.g., CdSe, CdS, and CdS or CdSe coated with ZnS) and magnetic (e.g., ferromagnetite) colloidal materials have been described. Other particle types include ZnS, ZnO, TiO2, AgI, AgBr, HgI2, PbS, PbSe, ZnTe, CdTe, In2 S3, In2 Se3, Cd3 P2, Cd3 As2, InAs, and GaAs.
Mirkin et al. (U.S. Pat. No. 6,361,944) describes aggregation assays for detecting a nucleic acid in a sample. This method involves incubating a sample thought to contain a nucleic acid with particles having oligonucleotides attached to the surface (particle-oligonucleotide conjugates). The oligonucleotides on each particle have an oligonucleotide complementary to one of the sequences of at least two portions of the nucleic acid. Alternatively, at least two types of particles having oligonucleotides attached may be used. The oligonucleotides on the first type of particles having a sequence complementary to one portion of the nucleic acid and the oligonucleotides on the second type of particles have a sequence complementary to a second portion of the sequence of the nucleic acid. The incubation takes place under conditions effective to allow hybridization of the oligonucleotides on the particles with the nucleic acid in the sample. Such a hybridization results in an aggregation of the particles. This produces a color change which may be detected visually or with simple instrumentation. For example, the aggregation of gold particles results in a color change from red to purple (Mirkin (U.S. Pat. No. 6,361,944)).
Methods of detection based on observing such a color change with the naked eye are cheap, fast, simple, robust (the reagents are stable) and do not require specialized or expensive equipment. This makes such methods particularly suitable for use in applications such as the detection of lead in paint or heavy metals in water.